PPARα activation promotes liver progenitor cell-mediated liver regeneration by suppressing YAP signaling in zebrafish

Despite the robust regenerative capacity of the liver, prolonged and severe liver damage impairs liver regeneration, leading to liver failure. Since the liver co-opts the differentiation of liver progenitor cells (LPCs) into hepatocytes to restore functional hepatocytes, augmenting LPC-mediated liver regeneration may be beneficial to patients with chronic liver diseases. However, the molecular mechanisms underlying LPC-to-hepatocyte differentiation have remained largely unknown. Using the zebrafish model of LPC-mediated liver regeneration, Tg(fabp10a:pt-β-catenin), we present that peroxisome proliferator-activated receptor-alpha (PPARα) activation augments LPC-to-hepatocyte differentiation. We found that treating Tg(fabp10a:pt-β-catenin) larvae with GW7647, a potent PPARα agonist, enhanced the expression of hepatocyte markers and simultaneously reduced the expression of biliary epithelial cell (BEC)/LPC markers in the regenerating livers, indicating enhanced LPC-to-hepatocyte differentiation. Mechanistically, PPARα activation augments the differentiation by suppressing YAP signaling. The differentiation phenotypes resulting from GW7647 treatment were rescued by expressing a constitutively active form of Yap1. Moreover, we found that suppression of YAP signaling was sufficient to promote LPC-to-hepatocyte differentiation. Treating Tg(fabp10a:pt-β-catenin) larvae with the TEAD inhibitor K-975, which suppresses YAP signaling, phenocopied the effect of GW7647 on LPC differentiation. Altogether, our findings provide insights into augmenting LPC-mediated liver regeneration as a regenerative therapy for chronic liver diseases.

www.nature.com/scientificreports/Moreover, PPARα promotes cardiomyocyte maturation by enhancing Yap1 expression 22 .In addition to this crosstalk with YAP signaling, PPARα interacts with Notch and WNT signaling.PPARα controls vasculoprotection and vascular remodeling by enhancing Notch signaling through fatty acid oxidation (FAO) in endothelial cells 23 .PPARα also protects diabetic kidneys from renal fibrosis by blocking WNT signaling in renal proximal tubules 24 .
We recently reported that farnesoid X receptor (FXR) suppresses LPC-to-hepatocyte differentiation in zebrafish 15 .Given that FXR and PPARα play opposite roles in multiple processes in the liver, including FAO, gluconeogenesis, and autophagy 20 , we hypothesized that PPARα activation could promote LPC-to-hepatocyte differentiation.By testing this hypothesis, we here present that PPARα activation indeed promotes LPC-tohepatocyte differentiation by suppressing YAP signaling.
We next further explored the effect of PPARα activation on LPC-to-hepatocyte differentiation.Section in situ hybridization analysis revealed the enhanced expression of hepatocyte marker genes (cyp7a1, gc, serpina1) and the reduced expression of the BEC marker epcam in GW7647-treated Tg(fabp10a:pt-β-catenin) livers (Fig. 1E), consistent with the qRT-PCR data.Moreover, we examined the formation of bile canaliculi by assessing the expression of Abcb11b, a bile salt export pump in the bile canaliculi of hepatocytes 28 .The number of Abcb11b + canaliculi per liver area was significantly increased in GW7647-treated Tg(fabp10a:pt-β-catenin) livers (Fig. 1F), suggesting promoted LPC-to-hepatocyte differentiation, with the caveat that the increased number could be due to reduced hepatocyte size.Altogether, these data indicate that PPARα activation promotes LPC-to-hepatocyte differentiation.
The pathway analysis also revealed that lipid metabolism-related pathways were enhanced in GW7647-treated Tg(fabp10a:pt-β-catenin) livers (Fig. 2C), as confirmed by qRT-PCR analysis (Fig. S1A).Given that FAO is one of the main target pathways of PPARα 20 (Fig. 2C) and that FAO regulates cell differentiation in injured kidneys 31 and vessels 23 , we tested if PPARα activation promotes LPC-to-hepatocyte differentiation by enhancing FAO.To suppress FAO, we used Etomoxir that inhibits Cpt1a 32 , the rate-limiting enzyme in FAO.However, Etomoxir treatment did not diminish the effect of PPARα activation on LPC-to-hepatocyte differentiation in Tg(fabp10a:ptβ-catenin) larvae (Fig. S1C).In addition, ppargc1a, a co-activator of PPARα in the induction of FAO-related genes 33 , mutant fish exhibited the same effect of GW7647 on LPC differentiation as their wild-type siblings (Fig. S1D).To further explore candidate pathways that mediate the effect of PPARα activation, we examined the expression levels of genes implicated in fate conversion between hepatocytes and BECs from the RNA-sequencing data.Intriguingly, we found that YAP target genes, ccn1 and ccn2, were downregulated in GW7647-treated Tg(fabp10a:pt-β-catenin) livers (Fig. 2A).Using a YAP reporter line, Tg(hCCN2:GFP) 34 , we also found that YAP activity was highly enhanced in Tg(fabp10a:pt-β-catenin) livers and that the activity was present in most LPCs, which express Anxa4, an LPC/BEC marker (Fig. 2D).Given a high YAP activity in BECs and ductular reactions 35 , it is possible that the reduced YAP signaling in GW7647-treated Tg(fabp10a:pt-β-catenin) livers is simply the consequence of enhanced LPC-to-hepatocyte differentiation rather than the consequence of PPARα activation.To exclude this possibility, we examined the expression of ccn1 and ccn2 in Tg(fabp10a:pt-β-catenin) larvae  www.nature.com/scientificreports/treated with AG1478, an EGFR inhibitor, known to promote LPC-to-hepatocyte differentiation 14 .qRT-PCR analysis showed that AG1478 treatment enhanced the expression of hepatocyte marker genes (cyp7a1, gc, ces2, serpina1) but not YAP target genes (ccn1 and ccn2) (Fig. S2A, B), supporting that PPARα activation suppresses YAP signaling in Tg(fabp10a:pt-β-catenin) livers.We also validated the reduced YAP signaling identified from RNA-sequencing data with qRT-PCR and Yap1 immunohistochemistry (Fig. 2E, F).Altogether, these findings reveal that PPARα activation suppresses YAP signaling in Tg(fabp10a:pt-β-catenin) livers.

PPARα activation promotes LPC-to-hepatocyte differentiation in the adult hepatocyte ablation model as well
We tested the effect of GW7647 on LPC-to-hepatocyte differentiation in another model of LPC-mediated liver regeneration in which liver regeneration is not caused by β-catenin dysregulation: the hepatocyte ablation model with Tg(fabp10a:CFP-NTR) fish that express nitroreductase (NTR) specifically in hepatocytes 13 .NTR converts metronidazole (Mtz) into a cytotoxic drug; thus, treating Tg(fabp10a:CFP-NTR) fish with Mtz ablates hepatocytes that express NTR.In this model, upon massive hepatocyte ablation, BECs first dedifferentiate into LPCs, and the LPCs subsequently differentiate into hepatocytes 13 .Tg(fabp10a:CFP-NTR) male adults were treated with Mtz for 5 h and subsequently with GW7647 from R1d to R3d for 48 h, and their livers were harvested at R3d for qRT-PCR analysis (Fig. S4A).Although its effects were weaker than those in the Tg(fabp10a:pt-β-catenin) model, GW7647 treatment increased the expression of two hepatocyte marker genes (fabp10a, gc) and decreased the expression of the Yap target genes (ccn1, ccn2) (Fig. S4B), suggesting the enhanced LPC-to-hepatocyte differentiation.

Discussion
In this study, we present that PPARα activation promotes LPC-to-hepatocyte differentiation by suppressing YAP signaling in LPCs.Although it has been reported that PPARα regulates hepatocyte proliferation-mediated liver regeneration 21,42,43 , it is elusive whether PPARα also regulates cell fate conversion-mediated liver regeneration, particularly LPC-to-hepatocyte differentiation.Previous in vitro studies 44,45 produced a discrepancy in the role of PPARα in LPC-to-hepatocyte differentiation.Here, using the zebrafish in vivo model, we demonstrate that PPARα activation promotes LPC-to-hepatocyte differentiation.Furthermore, we present the mechanism by which PPARα activation promotes the differentiation: suppressing YAP signaling in LPCs.Given the positive relationship between PPARα and YAP signaling in hepatocytes 21 and cardiomyocytes 22 , our finding about their relationship in LPCs is rather surprising.However, our rescue experiments with enhanced YAP signaling (Fig. 4) prove that PPARα activation promotes LPC-to-hepatocyte differentiation by suppressing YAP signaling.It was recently reported that PPARα interacts with YAP signaling in the liver 21 and heart 22 .Upon PHx, PPARα activation enhances its physical interaction with YAP1 in hepatocytes, thereby facilitating the translocation of YAP1 into the nucleus and subsequent hepatocyte proliferation 21 .During heart development, PPARα enhances Yap1 expression by binding its promoter, thereby driving cardiomyocyte maturation 22 .In both cases, PPARα positively regulates YAP signaling.We also observed in zebrafish that PPARα activation enhanced YAP signaling in the normal liver (Fig. S3).By contrast, we present that PPARα activation negatively regulates YAP signaling in LPCs.These opposite relationships between PPARα and YAP signaling imply that transcriptional regulators could have context-dependent, distinct roles.For example, another nuclear receptor, FXR, promotes hepatocyte proliferation following PHx 46,47 , whereas it suppresses LPC proliferation in BEC-driven liver regeneration 15 .Different contexts may make PPARα have distinct, or sometimes opposite, effects on YAP signaling.
YAP signaling is one of the crucial signaling pathways in hepatobiliary plasticity 48 .It positively controls hepatocyte-to-BEC conversion and LPC activation, also known as ductular reactions.Hepatocyte-specific overactivation of YAP signaling induced hepatocyte-to-BEC conversion 38,[49][50][51] , whereas hepatocyte-specific suppression of YAP signaling reduced ductular reactions 35,52 and BEC marker expression in hepatocytes 35,52,53 .Given this role in hepatocyte-to-BEC conversion, it can be assumed that suppressing YAP signaling may promote BECto-hepatocyte conversion or LPC-to-hepatocyte differentiation.Indeed, in vitro experiments showed that suppressing YAP signaling promoted hepatocyte differentiation in LPC lines 54 and induced hepatocyte-like cells 55 ; however, such in vivo evidence has been lacking.Using two zebrafish models of LPC-mediated liver regeneration, Tg(fabp10a:pt-β-catenin) and Tg(fabp10a:CFP-NTR), we hereby present in vivo evidence.In the adult hepatocyte ablation model, we observed rather subtle differences in the expression of hepatocyte markers and Yap1 target genes between DMSO-and GW7647-treated groups compared with those in the Tg(fabp10a:pt-β-catenin) model.We surmise that these variations could stem from different cellular origins of LPCs between the two models.Specifically, in the Tg(fabp10a:pt-β-catenin) model, ~ 70% and ~ 30% of LPCs originate from hepatocytes and BECs, respectively 14 , whereas in the adult hepatocyte ablation model, nearly all LPCs originate from BECs 56 .Given that hepatocyte-derived LPCs are distinct from BEC-derived LPCs in gene expression profiles 57 , we speculate that upon GW7647 treatment, hepatocyte-derived LPCs might differentiate into hepatocytes better than BEC-derived LPCs.
In summary, we provide the in vivo evidence that suppression of YAP signaling promotes LPC-to-hepatocyte differentiation.Furthermore, we provide a positive role for PPARα in LPC-to-hepatocyte differentiation.Our study suggests PPARα agonists as potential drugs that cannot only reduce a hepatic lipid level but also promote LPC-mediated liver regeneration.

Zebrafish lines
Zebrafish (Danio rerio) were maintained at 28.5 °C on a 14 h light/10 h dark cycle.Embryos and adult fish were raised and maintained under standard laboratory conditions 58 .We used the ppargc1a sa34243 mutant and the following transgenic lines: Tg(fabp10a:pt-β-catenin) s704 , Tg(fabp10a:CFP-NTR) s931 , Tg(hs:cayap1) zf622 , and Tg(hCCN2:GFP) ia48 .Their full names and references are listed in Table S1.All protocols used within this study were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh School of Medicine and conform to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.The reporting in this manuscript follows the recommendations in the ARRIVE guidelines 59 .For all experiments, we used embryos from multiple clutches; our breeding strategy is mass breeding to put 3-4 males and 5-6 females in the same breeding tank.We drew conclusions following at least three independent experiments.

The adult hepatocyte ablation model, Tg(fabp10a:CFP-NTR)
Six month old male Tg(fabp10a:CFP-NTR) fish were treated with 5 mM Mtz in system water containing 0.5% DMSO for 5 h to ablate hepatocytes.24 h after Mtz washout (R1d), the fish were treated with 1 mM GW7647 for 48 h and harvested at R3d.

qRT-PCR
Total RNA was extracted from 30 to 40 dissected livers for each condition using the RNeasy Micro Kit (Qiagen, Hilden, Germany); cDNA was synthesized from the RNA using the SuperScript ® III First-Strand Synthesis SuperMix (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocols.qRT-PCR was performed as previously described 14 using the QuantStudio 12K Flex machine (Applied Biosystems, Waltham, MA) with the iTaqTM Universal SYBR ® Green Supermix (Bio-Rad, Hercules, CA).eef1a1l1 was used for normalization as previously described 60 .At least three independent experiments were performed.For each replicate with larval livers, the control value was first set to 1, and then the values of the experimental groups were calculated relative to the control value.The primers used for qRT-PCR are listed in Table S3; the Ct values of qRT-PCR data are listed in Table S4.

RNA-sequencing
Livers were dissected from 30 to 40 DMSO-and GW7647-treated larvae.For adult experiments, the individual liver was dissected without pooling.Total RNA was extracted from the dissected livers using RNeasy Micro kit (Qiagen, Hilden, Germany) and sent to the University of Pittsburgh Genomics Core for library preparation and sequencing.Library preparation was performed using the TruSEQ Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA) according to the manufacturer's instructions.Sequencing was performed on a NextSeq 500 (Illumina, San Diego, CA) for 2 × 75-bp paired-end reads.These data have been deposited in NCBI's Gene Expression Omnibus (GSE226923).

RNA-sequencing analysis
Raw sequencing data were imported into CLC Genomics Workbench, and reads were mapped to the zebrafish reference genome.Differentially expressed genes (DEGs) used in the pathway analysis were determined between DMSO-and GW7647-treated groups using filters to select genes with Expr Fold Change ≥ |1.5| and Expr False Discovery Rate ≤ 0.05.DEGs were imported into Ingenuity Pathway Analysis to identify signaling pathways and upstream regulators affected by PPARα activation.Section in situ hybridization was performed as previously described 14 .cDNA from 14-or 15-dpf larvae was used as a template for PCR to amplify genes of interest; PCR products were used for in situ probe synthesis.The primers used for the probe synthesis are listed in Table S2.In the case of gc 61 and epcam 62 , their probes were synthesized using plasmids containing the genes.Immunostaining was performed as previously described 14 , using the following antibodies: mouse anti-Bhmt (1:500; a gift from Jinrong Peng at Zhejiang University), mouse anti-Anxa4 (1:300; Abcam, Cambridge, UK), rabbit anti-Abcb11 (1:800; Kamiya Biomedical, Seattle, WA), and Alexa Fluor 488-, 568-, and 647-conjugated secondary antibodies (1:500; Thermo Fisher Scientific, Waltham, MA).For section, larvae were fixed with 4% paraformaldehyde/PBS, embedded in Tissue Freezing Medium (Ted Fella, Redding, CA), and cryo-sectioned to 10-µm thickness.

Yap1 immunohistochemistry
Zebrafish larvae were fixed with Dietrich's fixative (3.7% formaldehyde/2% glacial acetic acid/30% ethanol) at room temperature for 24 h and processed for paraffin embedding.Paraffin block was prepared as previously described 63 .Samples were cut into 5-µm sections, and the sectioned samples were microwaved for 12 min in pH 6.0 sodium citrate buffer for antigen retrieval.After cooling, samples were placed in 3% H 2 O 2 for 10 min to quench endogenous peroxide activity.After washing with PBS, slides were blocked with Super Block (ScyTek Laboratories, Logan, UT) for 10 min.Samples were incubated with rabbit anti-Yap1 antibodies (1:00; Cell Signaling, Danvers, MA) and then with biotinylated secondary antibodies (1:500; EMD Millipore, Darmstadt, Germany) for 15 min at room temperature.After washing with PBS, samples proceeded with Vectastain ABC Elite kit (Vector Laboratories, Newark, CA), and the signal was developed with DAB Peroxidase Substrate Kit (Vector Laboratories, Newark, CA).Slides were counterstained with hematoxylin (Thermo Fisher Scientific, Newark, CA) and dehydrated to xylene, and coverslips were applied with Cytoseal XYL (Thermo Fisher Scientific, Newark, CA).

Heat-shock condition
Tg(hs:cayap1) larvae were heat-shocked at 12 or 13 dpf 6 h before compound treatments.The larvae were transferred into egg water pre-warmed to 38.5 °C and kept at 38.5 °C for 30 min, as previously described 64 .